Bone marrow-derived endothelial progenitor cells (EPCs) hold great promise in regenerative medicine for the treatment of chronic or currently incurable cardiovascular diseases. The homing of EPCs to neovascular areas requires a coordinated sequence of multistep events, including chemoattraction, adhesion, and invasion, before differentiation into mature endothelial cells and incorporation into active neovasculature. Impaired EPC function in mobilization and homing affects vascular homeostasis and also compensatory angiogenesis. Circulating EPCs may thus provide an endogenous repair mechanism to counteract ongoing risk factor-induced endothelial injury and to replace dysfunctional endothelium. Members of the SMYD protein family represent an emerging group of lysine methyltransferases that are particularly abundant in the cytoplasm, with SMYD1 being most highly expressed in heart and skeletal muscles. SMYD1 knockout in mice results in early embryonic lethality due to disruption of cardiac differentiation and morphogenesis. It has been reported that the chemokine receptor CXCR2 and its cognate ligands mediate EPC recruitment and angiogenesis in endothelial injury and myocardial ischemia. Compared with what is known about CXCR2 phosphorylation and other posttranslational modifications (such as ubiquitination and glycosylation), nothing is known about the lysine methylation of CXCR2, and how CXCR2 methylation might influence CXCR2 activation and signaling, especially as involved in EPC biology. The long-term goal is to better understand the molecular mechanisms regulating EPC homing and angiogenesis, in the hope of revealing novel therapeutic targets for certain vascular diseases. The hypothesis of this application is that lysine methylation of CXCR2 by the histone methyltransferase SMYD1 regulates EPC homing and angiogenesis through modulating CXCR2 activation and signaling. The specific aims of this project are: first, to examine the effect of SMYD1 in modulating CXCR2- mediated EPC migratory and angiogenic activities; and second, to characterize the molecular mechanism by which SMYD1 regulates CXCR2-mediated EPC function. A combination of multiple approaches, i.e., molecular and biochemical techniques, cellular functional assays, and in vivo animal models, will be used to test the hypothesis. The proposed research is innovative because by exploring a previously unrecognized lysine methylation of CXCR2 in modulating EPC functions, new potential therapeutic paradigms may evolve for EPC- based cell therapy in aberrant angiogenesis. In addition, this is the first study to investigate methylation of CXCR2 that may be the first non-histone target/substrate of the histone lysine methyltransferase SMYD1. The anticipated results from this proposal are the identification of a yet-unexplored role of the lysine methyltransferase SMYD1 in regulating CXCR2 function and the establishment of a novel link between lysine methylation, chemokine receptor activation, and endothelial signaling during vascular development and injury. Such results are expected to have an important impact in EPC biology, because the mechanistic characterization of the SMYD1-catalyzed CXCR2 lysine methylation are likely to provide valuable information for enhancing EPC-based cell therapy for certain vascular diseases, as well as to fundamentally advance the field of chemokine receptor biology.